The Screening And Identification Of Immune-related Genes And Regulatory Factors In Mongolian Horse

Mongolian Horse is one of the most ancient horse breeds in the world,being known as a Chinese native horse,which is characterized as disease-resistance,cold-resistance and crude feed tolerance.Analysis of immune-related genes provides insight into the genetic characteristics of disease-resistance in Mongolian Horses,which presents not only the theoretical basis for the research working in Mongolian Horse disease-resistance,but also the positive effect on protecting the germplasm resources of Mongolian Horse.RNA-Seq technology was used in this study to perform mRNA and IncRNA sequencing analysis on Mongolian Horse and Thoroughbred.Based on the sequencing data,the immune-related differentially expressed genes were screened.Secondly,the expression of the immune-related differentially expressed genes in the immune tissue was analyzed at the RNA level and the protein level.Results were as follows:1.In this study,six RNA libraries were constructed.Totally,996 M(million)raw reads(PE:2x 150 bp),and about 882 M clean reads were obtained after quality control for each library.Average 98%of clean reads can be mapped to the Thoroughbred reference genome.2.A total of 18,783 mRNAs were identified from 6 libraries,of which 1199 mRNAs were significantly differentially expressed between the two breeds(460 for up-regulated mRNA and 739 for down-regulated mRNA).The GO function enrichment analysis of differentially expressed genes revealed that the significantly enriched GO pathways of the two groups of proteins were mainly involved in immune responses,including inflammatory response,immune system processes,defense responses,and responses to stimuli.KEGG is significantly enriched in important immune-related pathways such as NF-?B signaling pathway and TNF signaling pathway.3.A total of 3473 novel IncRNA were identified from 6 libraries.These novel IncRNAs characterize few exon numbers,short transcript length and low expression abundance,but strong tissue specificity.Differential expression analysis revealed that 790 IncRNAs were identified to have significant difference between the two breeds(563 IncRNAs up-regulated and 227 IncRNAs down-regulated).There were 1062 target genes of IncRNA significantly differentially expressed,of which 203 were related to immunity.The KEGG pathway enrichment analysis revealed that 6 pathways reached significant Levels(P<0.05),and the pathways of most enriched of target genes were viral infection pathways.4.qRT-PCR was used to verify the accuracy in differential expression of NF-?B pathway-related genes in blood,spleen,liver and lung tissues in Mongolian Horse and Thoroughbred.The results showed that the expression of the selected 8 genes in the blood,spleen and liver was lower in Mongolian Horse,while the expression level of lungs is higher than that in Thoroughbred.At the same time,the differential expression analysis of NF-?B protein and IL-1? protein by Western blot showed that the expression levels of NF-?B protein and IL-1? protein in lungs were higher in Mongolian Horse than Thoroughbred.